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August 10, 2016

GCN5 for yeast is a bit like a complete English breakfast for people from the US —nonessential. Image from Wikimedia Commons.

A 2013 poll identified the top 20 modern necessities British people couldn’t live without. Some we can all relate to like smartphones, daily showers, and the internet, while others are more British-specific like a cup of tea or a full English breakfast.

Of course none of these are true necessities like food, water or air. We wouldn’t be as happy, nor as competitive, without some of these modern necessities, but we’d obviously still be alive. (But is life without the Internet really living?)

It turns out that the GCN5 gene is more like water or air for most eukaryotes—they can’t live without it. But our old friend Saccharomyces cerevisiae is different. This yeast isn’t as happy without GCN5, but it soldiers on nonetheless.

This nonessentiality, combined with its powerful genetics, makes yeast a great system for exploring what GCN5 does. And this is just what Petty and coworkers did with this important member of the histone acetyltransferase (HAT) family in a new study in GENETICS.

GCN5, like other HATs, transfers an acetyl group to histones, which results in increased activity of nearby genes. Consistent with this, previous work has shown that GCN5 acetylates histone H3 in the promoters of active genes.

HATs, along with their countervailing proteins histone deacetylases, as well as kinases, phosphatases, methyltransferases and so on, all work together to change gene expression on the fly in response to all sorts of different stimuli. These can include environmental signals, entering the cell cycle, or whatever.

Understanding how HATs work is critical for understanding how we (and other beasts) change gene expression in response to these signals. Which is what makes GCN5 in yeast such a great system. A strain deleted for GCN5 is sick, but alive, so we can study what happens when it is gone. And we can explore what we can do to fix its problems.

One of the many problems that yeast lacking GCN5 have is that they grow more poorly at high temperature than do yeast with GCN5. These researchers took advantage of this and looked for high copy suppressors of this temperature sensitivity. They found multiple genes, but the most common was RTS1, one of two regulatory subunits of the PP2A phosphatase complex.

Deleting GCN5 causes more problems than temperature-sensitivity, and overexpressing RTS1 restored some, but not all, of them. For example, RTS1 overexpression helped make the Δgcn5 strain less sensitive to DNA damage, less susceptible to microtubule disruption, better able to grow on nonfermentable carbon sources like glycerol or ethanol, and more able to progress into S phase during mitosis. But lots of PP2A^Rts1 could not rescue the abnormal buds nor the sporulation problems seen in a diploid lacking GCN5.

When the researchers deleted RTS1 or some of the genes that code for other critical components of the PP2A^Rts1 complex in a Δgcn5 strain, the strain died. The same was not true of a second regulatory subunit that can be part of the PP2A complex, CDC55—its deletion was not lethal, nor did it rescue the temperature sensitivity of the Δgcn5 strain.

Petty and coworkers provided evidence that the phosphatase activity of the PP2A^Rts1 complex was important by showing that okadaic acid, an inhibitor of this family of phosphatases, prevented Rts1p from rescuing the Δgcn5 strain’s temperature sensitivity. The easiest explanation is that the rescue happens because of the phosphatase activity of PP2A^Rts1, but it is also possible that a different member of the family might be providing the phosphatase activity.

So it looks like there is something important happening between PP2A and GCN5. Petty and coworkers next set out to find out what that might be.

First they showed that deleting GCN5 causes a decrease in the levels of core histones in the cell and that RTS1 overexpression fixed this problem. This happened at the transcription level as the RNA levels of the yeast histone genes (with the exception of HTA1) all showed reduced expression in the absence of GCN5, which again was restored when RTS1 was overexpressed.

Histone genes are normally turned on at the end of G1, then shut off at the end of S phase. This makes sense, as a cell needs to make more histones when it makes a new copy of its genome and this happens during S phase!

What might be essential in one setting is not necessarily so in another. Image from Wikimedia Commons.

Consistent with the reduced histone gene expression seen earlier, the Δgcn5 strain failed to increase histone gene activity at the end of G1. Overexpressing RTS1 restored this induction. So it looks like GCN5 is involved in turning on all the histone genes, except maybe HTA1, at the right time, and that RTS1 can compensate if there is a lot of it around.

As a final set of experiments, the authors looked at what PP2A^Rts1 might be doing to rescue histone gene expression when GCN5 was deleted. They decided to look at histone modifications.

For this they used the SHIMA (Scanning HIstone Mutagenesis with Alanine) library, in which all key serine and threonine residues were individually mutagenized to alanine. Even though PP2A^Rts1 is thought to be primarily a serine/threonine phosphatase, they also looked at three tyrosine residues (Y40, Y43, and Y45) on H2B by mutating each individually to phenylalanine.

They found that two residues on histone H2B, Y40 and T91, were required for RTS1 to be able to rescue the temperature sensitivity of a Δgcn5 strain. And mimicking the permanent phosphorylation of T91, by mutating it to either aspartic or glutamic acid, slowed the growth of wild type yeast and killed the deletion strain.

This tells us a lot about what GCN5 is doing in yeast, and it might also help us better understand certain human cancers. Turns out that the residue equivalent to T91 in mammals is phosphorylated in these cancers.

Petty and coworkers were able to learn all of this because of yeast’s powerful genetic tools, and because GCN5 is not essential in yeast. Once again, the awesome power of yeast genetics (#APOYG!) can help us understand human cell biology.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight Yeast and Human Disease